Chip-based flow cytometer type systems for analyzing fluorescently tagged particles

ABSTRACT

Portable systems for processing and analyzing biological or environmental samples as well as different configurations of chip-based flow cytometers are provided. The portable systems include an automated assay preparation module configured to process a sample into a fluid assay with fluorescently tagged particles and a microfluidic analysis module coupled to the fluid assay module, wherein the microfluidic analysis module includes a chip-based flow cytometer.

This application is a continuation of U.S. patent application Ser. No.11/750,000, filed May 17, 2007, which is now U.S. Pat. No. 9,810,707,which claims priority to U.S. Provisional Patent Application, Ser. No.60/747,483, filed May 17, 2006, the entire contents of which areincorporated herein by reference.

BACKGROUND OF THE INVENTION 1. Field of the Invention

The present invention generally relates to systems for processing andanalyzing fluid samples. Certain embodiments relate to chip based flowcytometer type systems configured to perform measurements of samplesincluding fluorescently tagged particles.

2. Description of the Related Art

The following description and examples are not admitted to be prior artby virtue of their inclusion in this section.

Generally, flow cytometers are devices configured to use opticaltechniques to measure one or more characteristics of particles or cellsin a fluid flowing through the devices. This particle or cellinterrogation can be exploited for a number of purposes such as assayingfor various chemical and biological molecules. Although flow cytometersoffer a number of advantages, the devices have a number ofdisadvantages. For example, flow cytometers generally utilize opticalcomponents that are sensitive to their environment. Furthermore, flowcytometers are typically time consuming to manufacture, complicated tooperate, and expensive. These characteristics often restrict the use offlow cytometers to highly trained technicians. Moreover, some samplesneed to be processed before being run through a flow cytometer and,therefore, significant laboratory resources and other equipment areoften needed for the analysis of assays.

It would, therefore, be advantageous to develop systems that measurecharacteristics of particles or cells in a fluid and are relativelysimple to operate. In addition, it would be beneficial for such systemsto be capable of performing the measurements without requiringsignificant laboratory resources for pre-processing of the samplefluids. Furthermore, it would be advantageous for the systems to includecomponents which are substantially insensitive to their environments.Moreover, it would be beneficial for such systems to be relativelyinexpensive and not time consuming to manufacture.

SUMMARY OF THE INVENTION

The following description of various embodiments of systems andchip-based flow cytometers are not to be construed in any way aslimiting the subject matter of the appended claims.

An embodiment of a system for processing and analyzing biological orenvironmental samples includes an automated assay preparation moduleconfigured to process a sample into a fluid assay with fluorescentlytagged particles and a microfluidic analysis module coupled to the

automated assay preparation module, wherein the microfluidic analysismodule includes a chip-based flow cytometer.

An embodiment of a system for analyzing a fluid sample includes achannel for routing a fluid sample with magnetic particles through thesystem and a means for inducing a magnetic field along at least aportion of the channel such that the magnetic particles flow within apredetermined region of the fluid sample.

Another embodiment of a system for analyzing a fluid sample includes achannel for routing a fluid sample having fluorescently tagged particlesthrough the system and an illumination subsystem including a lightsource system and an optical system collectively configured to directlight toward an interrogation region of the channel. The system furtherincludes a measurement subsystem with an aspherical mirror configured togather fluorescence emitted from the magnetic particles and anexamination system for analyzing the collected fluorescence.

An embodiment of a chip-based flow cytometer includes a first inputconduit for receiving a fluid sample with fluorescently taggedparticles, a second distinct conduit for receiving a sheath fluid, and afluid flow chamber coupled to the first and second input conduits. Thefluid flow chamber is configured to generate a fluid stream with thesample fluid confined within the sheath fluid and having a firstdimension of up to approximately 80 microns in a vertical directionperpendicular to the flow of the fluid stream and a second dimension ofup to approximately 25 microns in a horizontal direction perpendicularto the flow of the fluid stream.

Another embodiment of a chip-based flow cytometer includes a channel forrouting a fluid sample having fluorescently tagged particles through theflow cytometer. In addition, the chip-based flow cytometer includes anillumination subsystem with a light source system and an optical systemcollectively configured such that individual light sources within thelight source system direct light toward different spots within theinterrogation region. Moreover, the chip-based flow cytometer includes ameasurement subsystem having a collection system configured such thatthe fluorescent light emitted from the magnetic particles at each of thedifferent spots is collected by a different detector of the collectionsystem and an examination system for analyzing the collectedfluorescence.

BRIEF DESCRIPTION OF THE DRAWINGS

Further advantages of the present invention may become apparent to thoseskilled in the art with the benefit of the following detaileddescription of the preferred embodiments and upon reference to theaccompanying drawings in which:

FIG. 1 is a schematic diagram of a portable system configured to processand measure fluidic samples;

FIG. 2 is an isometric view of an exemplary embodiment of a microfluidicanalysis module that may be included in the portable system referencedin FIG. 1;

FIG. 3 is an isometric view of an exemplary embodiment of a fluidfocusing subsystem that may be included in the microfluidic analysismodule referenced in FIG. 2;

FIG. 4 is a side view of an exemplary collection of light sources,collection optics and detectors included in the microfluidic analysismodule referenced in FIG. 2;

FIG. 5 illustrates a cross-sectional view of an exemplary systemconfigured for preparing a fluid assay;

FIG. 6 illustrates a schematic drawing of a different exemplary systemconfigured for preparing a fluid assay;

FIG. 7 illustrates a prospective view of an exemplary system whichfollows the schematic layout of FIG. 6;

FIG. 8 illustrates a magnified prospective view of the reaction vesselincluded in the system depicted in FIG. 7;

FIG. 9 illustrates cross-sectional view of the reaction volume of thereaction vessel depicted in FIG. 8 during a series of process steps forpreparing a fluid assay;

FIG. 10 illustrates a magnified prospective view of the reagent packreceiver of the system depicted in FIG. 7 as well as a reagent pack;

FIG. 11A-C illustrates cross-sectional view of the reagent pack receiverdepicted in FIG. 10 with a reagent pack arranged therein and in avariety of positions to portray oscillation of the reagent pack;

FIG. 12 illustrates a flowchart of an exemplary method for preparing afluid assay;

FIG. 13 illustrates a flowchart of an exemplary method for processing afluid sample into a form that is compatible with a predetermined assay;

FIG. 14 illustrates a flowchart of an exemplary method for preparing anucleic acid assay;

FIG. 15 illustrates a flowchart of an exemplary method for preparing animmunoassay.

FIG. 16 is an isometric top view of an exemplary embodiment of areaction cartridge that may be included in the system embodimentsdescribed herein;

FIG. 17 illustrates different isometric views of another exemplaryembodiment of a reaction cartridge, at various times during operation ofthe reaction cartridge, that may be included in the system embodimentsdescribed herein; and

FIG. 18 is an isometric side view and a top view of a reagent storagemodule that may be included in the portable systems described herein.

While the invention is susceptible to various modifications andalternative forms, specific embodiments thereof are shown by way ofexample in the drawings and may herein be described in detail. Thedrawings may not be to scale. It should be understood, however, that thedrawings and detailed description thereto are not intended to limit theinvention to the particular form disclosed, but on the contrary, theintention is to cover all modifications, equivalents and alternativesfalling within the spirit and scope of the present invention as definedby the appended claims.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Turning now to the drawings, systems for processing and analyzing fluidsamples are provided. In general, the systems described herein may beused to process and/or analyze samples including, but not limited to,bodily fluids, environmental samples, and/or biological tissues andsubstances. Furthermore, as described in more detail below, the systemsdescribed herein are configured for processing samples withfluorescently tagged particles infused therein. In some embodiments, theparticles may be magnetic. It is noted that the figures are notnecessarily drawn to scale. In particular, the scale of some elements insome of the figures may be greatly exaggerated to emphasizecharacteristics of the elements. In addition, it is further noted thatthe figures are not drawn to the same scale. Elements shown in more thanone figure that may be similarly configured have been indicated usingthe same reference numerals.

FIG. 1 illustrates an exemplary portable system for processing andanalyzing fluidic samples. In general, the portable systems describedherein may include a processing module, an analysis module, and supportmodules including, but not limited to, fluidics, fluid storage, powersupply, computer hardware (and software), display, and a humaninterface. FIG. 1 illustrates portable system 12 opened showing anexemplary assortment and placement of modules within a base portion andlid of the system. It is noted, however, the portable systems describedherein are not necessarily so limited. In particular, other portablesystems having fewer or more modules than the ones described inreference to FIG. 1 may be considered. In addition or alternatively, oneor more of the modules described in reference to portable system 12 maybe arranged in a different position relative to the other modules. Forinstance, in some cases, portable system 12 may include differentmodules or no modules in the lid of the system. In other cases, portablesystem 12 may not include a lid. In any case, the “portability” of thesystems described herein may refer to systems being able to betransported by a user.

As shown in FIG. 1, portable system 12 may include computer 1, display2, battery 3, and human interface 9, all of which may include anysuitable component known in the art for the corresponding device. Insome cases, computer 1 may be used to convert measurements taken bymicrofluidic analysis module 5 (e.g., fluorescence measurements, lightscatter measurements, etc.) into values which reflect the amount ofanalyte present in a sample. In addition or alternatively, computer 1may be used to control the operations of other components withinportable system 12, such as but not limited to the components withinprocessing module 6 and microfluidic analysis module 5. In any case,display 2 may be used to present measurement values and/or convertedvalues to a user. In addition or alternative to the inclusion of battery3, portable system 12 may be equipped with an AC/DC converter. Humaninterface 9 may be configured such as to control the initiation ofprocessing or analyzing a fluid and/or manipulate the measurementsacquired during analysis. As further shown in FIG. 1, portable system 12may include reagent cartridge storage 4, processing module 6,microfluidic analysis module 5, sheath fluid module 7, and pump module8, each of which may be configured as described further herein. In someembodiments, the systems described herein may include one or moreinterchangeable modules that can be replaced such that the systems canbe used for different applications. Possible applications of theembodiments described herein include, but are not limited to, a medicaldiagnostic device or environmental biological sampler. The modularnature of the embodiments allows for easy and inexpensivereconfigurations from one application to another.

In some embodiments, microfluidic analysis module 5 may include a highlyintegrated chip-based flow cytometer. More specifically, microfluidicanalysis module 5 may include a fixed configuration of some or all ofthe following components fabricated as semiconductor devices:microfluidic channels, photo multiplier tubes, avalanche photodiodes,pin photo diodes, magnetic transducers, optical filters, lenses,mirrors, resonators, optical gain mediums, and excitation sources suchas light emitting diodes, resonant cavity light emitting diodes, diodelasers, vertical cavity surface emitting lasers, phosphorescentmaterials, radioactive materials, plasmas, and acoustic sources. Anexemplary configuration of components for microfluidic analysis module 5is illustrated in FIG. 2. In particular, FIG. 2 illustrates anembodiment of microfluidic analysis module 5 in which fluidic channels,fluidic focusing components, and collection optics are fixedly arrangedwithin semiconductor substrate 10. In some embodiments, microfluidicanalysis module 5 may also include light sources (e.g., light sources 14and 16 in FIG. 2) and optical components (e.g., beam splitter 18 in FIG.2) fixedly arranged relative to each other and semiconductor substrate10. In alternative cases, light sources 14 and 16 and/or beam splitter18 may be integrated within semiconductor substrate 10.

In general, the chip-based flow cytometer of microfluidic analysismodule 5 may be configured to measure one or more characteristics offluorescently tagged particles which are infused within a fluid flowingthrough the flow cytometer. More specifically, the chip-based flowcytometer may be configured to measure fluorescence emitted by particleswithin a sample and use the measurements to determine the presence of areagent associated with the particles. Based on the presence or absenceof the reagent, the system can determine the presence or absence of oneor more analytes in the samples and may also determine othercharacteristics of the one or more analytes such as concentration withinthe sample. In some cases, light scattering characteristics of theparticles may be additionally measured by the chip-based flow cytometerto determine the presence/absence of analyte within a sample and/orclassifications of the particles.

In general, the term “particle” as used herein may refer to anysubstrate used for the analysis of chemistry, biological, andenvironmental assays and may specifically refer to articles used toprovide and/or support molecular reactions for the qualification and/orquantification of an analyte of interest including but not limited tokinase activity. In addition, the term “particle” may reference articlesof a broad range of sizes, such as but not limited to articles havingdimensions between approximately 1 nm approximately 300 μm. Hence, theterm “particle” may refer to a number of different materials andconfigurations, including but not limited to particles, beads,polystyrene beads, microparticles, gold nanoparticles, quantum dots,nanodots, nanoparticles, composite particles (e.g., metal-polymericparticles or magnetite-polymeric particles), nanoshells, nanorods,nanotubes, microbeads, latex particles, latex beads, fluorescent beads,fluorescent particles, colored particles, colored beads, tissue, cells,micro-organisms, spores, organic matter, any non-organic matter, or anycombination thereof. Accordingly, any of such terms may beinterchangeable with the term “particle” used herein.

Recent innovations have allowed simultaneous analysis of multiple assaysthrough the use of distinguishable carrier particles. One example ofsuch an assay system is the xMAP® technology that is commerciallyavailable from Luminex Corporation of Austin, Tex. The xMAP technologyuses a family of dyed particles onto which one or more assay-specificreagents may be applied (e.g., by coupling to one or more functionalgroups on the surface of the particles). The particle platform employsdifferent sets of particles distinguishable by fluorescence. Forexample, the sets of particles may be distinguishable by wavelength offluorescence, intensity of fluorescence, ratio of intensities offluorescence at different wavelengths, etc. In general, the variation offluorescence may be integrated by different dyes and/or fluorophoresincorporated into the particles and/or are coupled to a surface of theparticles. In some embodiments, the sets of particles may beadditionally distinguishable by size and/or shape. In any case, aparticle platform having distinguishable carrier particles is generallyadvantageous because it uses fluid based kinetics to bind severaldifferent analytes to the assay-specific reagents. In particular, theparticles can be used to test for more than 100 different analytes in asample.

In general, each of the different sets of particles may have a differentreagent coupled to the particles. The different reagents may selectivelyreact with different analytes in the fluid sample. In other words, eachof the different reagents may react with one analyte in a sample, butmay not substantially react with any other analytes in the sample. Insome cases, one or more additional detectable reagents may be allowed toreact with one or more of the analytes. The one or more additionalreagents may be detectable (and possibly distinguishable) byfluorescence (e.g., wavelength of fluorescence, intensity offluorescence, etc.). In addition to the enhanced reaction kinetics, theuse of a multiplexed particle platform advantageously allows a user tosimply add or remove one or more subsets of particles, to or from thepopulation to which the sample is exposed, to change the tests beingperformed in a panel.

Turning back to FIG. 2, the operation of the flow cytometer may includeintroducing the sample on which an assay is being performed into inputconduit 22. Prior to being illuminated, the sample may, in someembodiments, be combined with a sheath fluid in fluid flow chamber 24 asshown in FIG. 2. The sheath fluid may be pumped by pump module 8 fromsheath fluid module 7, enter the analysis module through input conduit26, and may flow through sheath fluid channel 28 to fluid flow chamber24. Therefore, the sample and sheath fluids are injected into the flowcytometer through separate input ports. The two fluids meet in fluidflow chamber 24 and the chamber is configured such that a fluid streamis generated which includes the sheath fluid encompassing the samplefluid. An exemplary configuration of fluid flow chamber 24 isillustrated in FIG. 3. It is noted, however, that other fluid combiningtechniques may be used for the systems described herein including, butnot limited to, hydrodynamic focusing, electrokinetic focusing, acousticwave focusing, and magnetic focusing. Consequently, the systemsdescribed herein are not necessarily limited to the depictions of FIGS.2 and 3. For example, a fluid combining device that may be additionallyor alternatively used with the flow cytometer of microfluidic analysismodule 5 may be a hydrodynamic focusing cuvette arranged at a joinedinterface of channels 20 and 28. The cuvette may perform highly uniformfocusing and confinement of the sample stream in all directions that lieperpendicular to the axis of the cuvette.

As shown in FIG. 3, fluid flow chamber 24 may be configured to confinethe sample fluid within the sheath fluid in both the vertical andhorizontal directions. In this manner, the sheath fluid may surround thesample fluid from all sides. This physical confinement techniqueconfines the sample fluid to approximately the center of the streamgenerated from the fluid flow chamber 24 and, in some cases, narrows thesample fluid to a fraction of its previous diameter. By reducing thediameter of the sample fluid, particles in the flow are pulled fartherapart along the fluid channel allowing for easier interrogation. It hasbeen found, however, that the incorporation of fixed chip-basedcomponents within a flow cytometer offers notable measurement precisioneven with relatively wide fluid streams. Thus, fluid flow chamber 24 maybe configured to narrow a sample fluid diameter to a lesser degree thanin conventional systems. For instance, fluid flow chamber 24 may beconfigured to reduce the diameter of the sample fluid within channel 25to less than 10 times its diameter within channel 20 or, morespecifically, about 5 times its diameter within channel 20. Narrowingthe sample fluid to a lesser degree may advantageously reduce the amountof sheath fluid needed, reducing costs and waste.

Another manner to describe the confinement adaptations of fluid flowchamber 24 is to specify exemplary dimensions of the sample streamwithin the fluid stream it is configured to generate. For example, fluidflow chamber 24 may be configured such that the sample fluid has a firstdimension of up to approximately 80 microns in a vertical directionperpendicular to the flow of the fluid stream (i.e., a first dimensionof up to approximately 80 microns in the z-direction when flow of thefluid is in the x-direction). In addition, fluid flow chamber 24 may beconfigured such that the sample fluid has a second dimension of up toapproximately 25 microns in a horizontal direction perpendicular to theflow of the fluid stream (i.e., a second dimension of up toapproximately 25 microns in the y-direction when flow of the fluid is inthe x-direction). Larger or smaller dimensions for the sample fluid,however, may be considered as well, depending on the designspecifications of the flow cytometer.

In any case, the fluid stream generated from fluid flow chamber 24 mayflow through channel 25 as shown in FIGS. 2 and 3. More specifically,the fluid stream generated from fluid flow chamber 24 may flow to anoptical interrogation region of the flow cytometer, denoted ascollection optics 30 along channel 25 In FIG. 2. After passing throughthe optical interrogation region, the combined sample and sheath fluidsmay exit the analysis module through outlet 32. In some embodiments,fluid channel 25 may be configured to be non-removable. In other words,portable system [[25]]12 may include a fluid channel that is notdisposable. In contrast, the fluid channels in existing chip scaletechnologies are designed to be formed in a removable substrate so thatthe fluid channels may be disposable. Therefore, a distinguishingfeature of the embodiments described herein is that the fluid channelmay be neither removable nor disposable. In some embodiments, it may beadvantageous to arrange the sample fluid of the generated fluid streamin a predetermined location of fluid channel 25. More specifically, itmay be advantageous to have the particles within the sample fluidarranged in a relatively predictable location in channel 25 (e.g.,approximately the center of the fluid channel) such that the angles atwhich light may be directed toward the particles and emissions collectedfrom the particles for optimum operation of the flow cytometer may beanticipated.

In order to accommodate the arrangement of particles within apredetermined location of channel 25, the systems described herein may,in some embodiments, include means 27 for inducing a magnetic fieldalong at least a portion of channel 25 as shown in FIG. 2. For example,the flow cytometer of microfluidic analysis module 5 may include asolenoid coil wrapped around fluid channel 25. It is noted that thesystems described herein are not necessarily limited to such a magneticfield inducing mechanism. In particular, other means for inducing amagnetic field along at least a portion of channel 25 may beadditionally or alternatively used. In any case, means 27 may, in someembodiments, extend along the entirety of fluid channel 25 (i.e.,between fluid flow chamber 24 and output 32). In yet other embodiments,means 27 may extend along a limited section of fluid channel 25. Forexample, means 27 may, in some embodiments, be restricted to a portionof fluid channel 25 extending between fluid flow chamber 24 and throughthe interrogation region denoted by collection optics 30. In yet otherembodiments, means 27 may, in some embodiments, may be restricted to ashorter portion of fluid channel 25 such as in close proximity to andincluding the interrogation region. In any case, means 27 may beconfigured to generate a magnetic field around fluid channel 25 in someembodiments. Alternatively, means 27 may be configured to generate amagnetic field along less than the circumferential periphery of fluidchannel 25.

As noted above, the sample fluid surrounded by the sheath fluid flowsfrom fluid flow chamber 24 to the optical interrogation region of fluidchannel 25. In the optical interrogation region, light sources 14 and 16illuminate the sample fluid. Fluorescence emitted by the particleswithin the sample fluid is collected by collection optics 30 anddirected to one or more detectors of the flow cytometer, which may befixedly arranged relative to substrate 10 at an angle anticipated forthe emissions. Therefore, the optical interrogation region of fluidchannel 25 is defined by the locations along the fluid channel that areilluminated by the light beams and the locations along the fluid channelat which the collection optics collect light (e.g., fluorescent light,scattered light, or some combination thereof). As shown in FIG. 2, thelight from light sources 14 and 16 may be directed to differentlocations along fluid channel 25 that are spaced apart from each other.Such locations may be referred to herein as interrogation spots. Theinterrogation spots may include any suitable spacing, depending on thedesign specifications of the flow cytometer.

To facilitate multiple interrogation spots, the flow cytometer may, insome embodiments, include a beam splitter between one or more of lightsources 14 and 16 and fluid channel 25, such as shown with the inclusionof beam splitter 18 in FIG. 2. In general, beam splitter 18 may includeany suitable optical element/s that is configured to split light intotwo or more different beams of light. Thus, beam splitter 18 is notrestricted to the depiction of splitting light into three beams asillustrated in FIG. 2. Furthermore, the cytometer shown in FIG. 2 is notrestricted to using beam splitter 18 with respect to light source 14. Inparticular, the flow cytometer may additionally or alternatively includea beam splitter positioned to split light directed from light source 16.In some embodiments, it may be advantageous to include multiple lightsources within the flow cytometer, particularly one for eachinterrogation spot. As such, in some cases, the flow cytometer may notinclude beam splitters. In particular, due to the miniaturization ofdevice components within the chip-based flow cytometer, the detectors ofthe flow cytometer may need to collect full emissions from aninterrogation spot in order to adequately measure the fluorescence of aparticular channel. Consequently, in addition to including a differentlight source for each interrogation spot, the flow cytometer may includea different detector for each interrogation spot. Furthermore, the flowcytometer may include a different set of optical components, includingbut not limited to mirrors, lens, and/or filters, for each interrogationspot. An exemplary arrangement of light sources, optical elements, anddetectors used for the flow cytometers described herein is shown in FIG.4 and described in more detail below.

In any case, although FIG. 2 shows the inclusion two light sources withthe flow cytometer depicted therein, it is to be understood that theflow cytometer may include any number of light sources within itsillumination subsystem. Any suitable light sources may be employedwithin the flow cytometer, such as but not limited to light emittingdiodes (LEDs), lasers, arc lamps, fiber illuminators, light bulbs,incandescent lamps, or any other suitable light sources known in theart. In addition, the one or more light sources may be configured toemit monochromatic light, polychromatic light, broadband light, or somecombination thereof. Furthermore, light from the one or more lightsources may be directed to fluid channel 25 in any suitable direction(e.g., by suitable positioning of the light sources and/or by usingsuitable light directing optical elements). As such, although FIG. 2illustrates light sources 14 and 16 directing light toward fluid channel25 at substantially opposite directions, the systems described hereinare not necessarily so limited.

As noted above, FIG. 4 illustrates an exemplary arrangement of lightsources, optical elements, and detectors used for the flow cytometersdescribed herein. The exemplary arrangement includes four light sources38, which are substantially perpendicularly aligned with four distinctcollection optics 42 and four distinct detectors 44. In other words,collection optics 42 are arranged to collect light emitted from theparticles within the sample fluid along a direction that issubstantially perpendicular to a direction at which light from lightsources 38 is directed. Collection optics 42 may include one or morerefractive optical elements, each of which is configured to collectlight fluoresced and/or scattered from a corresponding location alongthe fluid channel. Detectors 44 may include any suitable detectors suchas but not limited to silicon avalanche photodiodes. In some cases, eachof light sources 38 may be configured to emit light having substantiallythe same wavelength. In other embodiments, however, one or more of lightsources 38 may be configured to emit light at a different wavelengththan the light emitted by other of light sources 38.

In some embodiments, the systems described herein may include filters 48positioned in between collection optics 42 and detectors 44. Filters 48may include any suitable optical elements known in the art such as butnot limited to spectral filters that are configured to limit thewavelength(s) of the light detected by detectors 44. As further shown inFIG. 4 the exemplary arrangement may include illumination optics 50.Illumination optics 50 may include a number of refractive opticalelements, which may include any suitable refractive optical elementsknown in the art. Each of the refractive optical elements ofillumination optics 50 may be configured to collimate one of light beams52 generated by light sources 38 of semiconductor light emitters. Inthis manner, the light beams may be directed to channel 25 alongdirections that are substantially parallel to each other. Although FIG.4 is shown to include four sets of components, it is to be understoodthat the systems described herein may include any suitable number ofcomponent sets.

FIG. 5 illustrates a different exemplary arrangement of light sources,optical elements, and detectors used for the flow cytometers describedherein which employs a configuration referred to herein as 90 degreeoff-axis illumination. In such configurations, the system may include anoptical element configured to direct light to fluid channel 25 at anangle that is about 90 degrees from the angle at which light sources 38are configured to direct light to the fluid channel. Alternatively,light sources 38 may be arranged to direct light at a 90 degree angle tofluid channel 25. More specifically, a mirror or other suitable opticalelement may be arranged such that light directed from light sources 38strikes fluid channel 25 at a direction that is substantiallyperpendicular to both the fluid channel and the optical stack thatincludes collection optics 42 and detectors 44 such that the 90° sidescatter component of the light is collected and detected for the opticalinterrogation method. To illustrate such a configuration, FIG. 5 depictsthe exemplary arrangement of components along an x-z plane, whereinfluid flow in channel 25 is in the x-direction and the position of theoptical elements and detectors are in the z-direction relative tochannel 25. In such embodiments, the light source/s is arranged in they-direction relative to channel 25 and, therefore, is not depicted inFIG. 5. Rather, light beam 54 directed from the light source/s is showncoming out of the page denoting the 90 degree off-axis illumination.Although FIG. 5 illustrates a single set of components, it is to beunderstood that the systems described herein may include any suitablenumber of distinct sets of components as similarly described for theconfiguration depicted in FIG. 4.

In some embodiments, the flow cytometers described herein may include anaspherical mirror for gathering the fluoresced and/or scattered light.In particular, an aspherical mirror may advantageously distort thecollected light to a level within a range of light which the detectorsmay be capable of detecting. More specifically, the detection range ofthe detectors cannot generally be scaled down in the same manner as theother devices in the chip-based flow cytometer and, thus, it may bebeneficial to incorporate an aspherical mirror to alter collected lightinto a range which is within the ability of the detectors. In someembodiments, the aspherical mirror may be coated with an anti-reflectivecoating (ARC) to aid in rejection of unwanted wavelengths, increasingthe signal-to-noise ratio of the collected light. In some embodiments,an ARC that is configured to reflect fluorescent emissions rather thanscattered light may be employed. In other embodiments, an ARC that isconfigured to reflect scattered light in addition to fluorescentemissions may be employed. Such configurations may be facilitated by thewavelength range the ARC is configured to reflect. As shown in FIG. 5,aspherical mirror 56 may be arranged on the side of fluid channel 25opposing collection optics 42, filter 48, and detector 44. In thismanner, aspherical mirror 56 may increase the amount of light collectedby the flow cytometer. It is noted that the configuration of componentsdescribed in reference to FIG. 4 may also include an aspherical mirror.

In response to the excitation, particles in the sample flow mayfluoresce thereby signaling the presence of at least one of thefollowing materials with the sample fluid including, but not limited to,particle identification dye, enzymes, proteins, amino acids, DNA, RNA,antibodies, and antigens. In addition to direct fluorescence being usedas an indicator, the presence of a magnetic field may be used to furthermodulate the excitation of the particle or emission from the particle.One advantageous feature of the analysis module is the ability tomeasure or “read” and classify fluorescent particles in real time usingoutput generated by multiple channels of the analysis module. In someembodiments, the system may be configured to perform the measurements bygating a response of particles to illumination as the sample flowsthrough the interrogation region in fluid channel 25. For example, asthe sample flows through the optical interrogation region of theanalysis module, gating the response of the particles to illuminationmay be performed by collecting and detecting the fluorescence and/orscattered light with the collection optics and the one or more detectorsand comparing the output of the one or more detectors to a predeterminedthreshold. If the output is above the predetermined threshold, thesystem may determine that a microsphere caused the fluorescence and/orscattered light and therefore that the microsphere is passing throughthe interrogation region. As such, the fluorescence and/or scatteredlight corresponding to the microsphere may be recorded or otherwiseprocessed by the system (e.g., processed for real-time classification).

In another embodiment, the system may be configured to perform themeasurements by distinguishing between single particles flowing throughthe interrogation region of fluid channel 25 and multiple particlesflowing through the interrogation region at substantially the same time.For example, scattered light detected by one or more detectors of theanalysis module may generally be proportional to the volume of theparticles that are illuminated by the one or more light sources.Therefore, output of the one or more detectors may be used to determinea diameter and/or volume of the particles that are in the opticalinterrogation region. In addition, the output of the one or moredetectors and the known size or volume of the particles may be used toidentify more than one microsphere that are stuck together or that arepassing through the optical interrogation region at approximately thesame time. Therefore, such particles may be distinguished from othersample particles and calibration particles. Furthermore, the output ofthe one or more detectors may be used to distinguish between sampleparticles and calibration particles if the sample particles andcalibration particles have different sizes.

As noted above, portable system 12 may, in some embodiments, includeprocessing module 6. Such a module may generally be configured toprocess samples before the samples are introduced into microfluidicanalysis module 5. The processing may include any one or more ofparticle size filtering, centrifuging, analyte isolation, analyteamplification, washing of the sample, cell lysing, clotting factorneutralization, pH regulation, temperature cycling, reagent mixing, andassay reaction. Other processing steps may be considered as well.Exemplary configurations of systems which may be used for processingmodule 6 are illustrated in FIGS. 6-9 and described in more detailbelow. Exemplary components which may be used to support such systemsare illustrated in FIGS. 10-11A-C and 16-18. In addition, methods foroperating such process modules are outlined in flowcharts of FIGS.12-15. In yet other embodiments, portable system 12 may not includeprocessing module 6. In such cases, portable system 12 may be configuredto analyze raw samples (i.e., samples which have not be pre-processed)or samples which have been pre-processed independent of portable system12.

FIG. 6 illustrates a schematic drawing of fluid assay preparation system51 and FIG. 7 illustrates a perspective view of an exemplaryconfiguration for fluid assay preparation system 51. As shown in FIGS. 6and 7, fluid assay preparation system 51 may include sample inlet 53 forintroducing a fluid sample into system 51. In some embodiments, fluidassay preparation system 51 may include sample preprocessing system 54for processing the sample prior to being introduced into inlet 53. Thepreprocessing system may be configured to perform any of the stepsdescribed below in reference to FIG. 13 or any state transformationsteps. For example, a wetted wall cyclone may be considered forcondensing a gas sample into a liquid. Fluid assay preparation system 51further includes reagent pack 56 including a plurality of vessels eachfilled with different reagents. Reagent pack 56 may also include one ormore vessels for receiving waste streams from the fluid assaypreparation performed by system 51. It is noted that the reagents notedin FIG. 6 are exemplary and fluid assay preparation system 51 is notnecessarily so limited. For processing a sample, reagent pack 56 may bearranged within reagent pack receiver 64 (shown in FIG. 7) and coupledto multi-port valve 58, which in turn may be coupled to reaction vessel60 by fluidic lines. In addition, input 53 may be coupled to multi-portvalve 58. In an alternative embodiment, fluid assay preparation system51 may include one or more individual valves respectively coupled to thevessels of reagent pack 56 and/or input 53. In any case, reagent pack 56may be stored within reagent storage module 4 of portable system 12prior to being used by processing module 6.

As shown in FIGS. 6 and 7, fluid assay preparation system 51 may furtherinclude control electronics 66 and pump 62 coupled to multi-port valve58. Alternatively, fluid assay preparation system 51 may use pump module8 of portable system 12. In either case, control electronics 66, pump 62or pump module 8, and multi-port valve 58 may be collectively configuredsuch that the sample introduced within input 53 and the reagents withinreagent pack 56 may be introduced into reaction vessel as well as drawntherefrom, preferably at separate stages within the fluid assaypreparation process. A more detailed description of exemplary routingsof the reagents to and from reaction vessel 60 is provided in referenceto FIGS. 12-15 below.

As shown in FIG. 7, fluid assay preparation system 51 may furtherinclude storage medium 68 coupled to control electronics 66. In general,storage medium 68 may include program instructions which are executableby a processor for automating the preparation of a fluid assay, such asbut not limited to the steps described in below in reference to theflowcharts depicted in FIGS. 12-15. The storage medium may include butis not limited to a read-only memory, a random access memory, a magneticor optical disk, or a magnetic tape. The program instructions may beimplemented in any of various ways, including procedure-basedtechniques, component-based techniques, and/or object-orientedtechniques, among others. For example, the program instructions may beimplemented using ActiveX controls, C++ objects, JavaBeans, MicrosoftFoundation Classes (“MFC”), or other technologies or methodologies, asdesired. In some embodiments, storage medium 246 may include a processorfor executing the program instructions. In other embodiments, however,storage medium 246 may be configured to be coupled to a processor (e.g.,by a transmission medium). In either case, the processor may takevarious forms, including a personal computer system, mainframe computersystem, workstation, network appliance, Internet appliance, personaldigital assistant (PDA), a digital signal processor (DSP), fieldprogrammable gate array (FPGA), or other device. In general, the term“computer system” may be broadly defined to encompass any device havingone or more processors, which executes instructions from a memorymedium. It is noted that storage medium 68 is shown coupled to controlelectronics 66 by dotted lines in FIG. 7 to indicate that the connectionmay be either fixed or detachable.

In some embodiments, fluid assay preparation system 51 may be configuredfor multiple use operation and, therefore, may be reusable. Inparticular, fluid assay preparation system 51 may be configured toprepare multiple fluid assays, including those of the same or differentmakeup. In other embodiments, fluid assay preparation system 51 may beconfigured for single use operations and, therefore, may be configuredto be disposable. In either case, reagent pack 56 may be configured forsingle or multiple use operations. As such, reagent pack 56 may beconfigured to be disposable (i.e., thrown away after a single fluidassay has been prepared) or may be reusable (i.e., includes reagentamounts sufficient to prepare multiple assays). In the latter case, thevessels of reagent pack 56 may be configured to be disposed after one ormore of the reagents are consumed or may be configured to be refilled.In either embodiment, reagent pack 56 allows for easy replacement andmay be generally inexpensive to maintain and produce.

An exemplary configuration for reaction vessel 60 is shown in FIG. 8. Asshown in FIG. 8, reaction vessel 60 may include an injection/aspirationneedle which may be used to receive and dispatch solutions to and fromreaction vessel 60, such as from/to reagent pack 56 and input 53 offluid assay preparation module 50. Reaction vessel 60 further includesan analysis module aspiration needle, which may be used to dispatchsolutions to microfludic analysis module 5. Reaction vessel 60 mayfurther include a vent port, magnet actuator, and magnet 70 as shown inFIG. 8. In some embodiments reaction vessel 60 may further include asonication system or, more generally, fluid assay preparation system 51may include a sonication system in proximity to reaction vessel 60. Anexemplary manner in which to utilize the magnet actuator and magnet forpreparing fluid assay is shown in FIG. 9. In particular, FIG. 9 includessnapshot I in which the reaction vessel volume is empty and, therefore,no fluid sample or reagent has been introduced therein. FIG. 9 furtherillustrates snapshot II in which the reaction vessel volume is filledwith a fluid sample, magnetic particles and, in some cases, one or morereagents. Snapshot III of FIG. 9 illustrates magnet 70 actuated inproximity to the reaction vessel volume to immobilize the magneticparticles and snapshot IV of FIG. 9 illustrates the reaction vesselvolume having the fluid removed when the magnetic particles areimmobilized. A more detailed description of possible stagings of such anoperation during a fluid assay preparation procedure is provided belowin reference to FIGS. 12-15.

FIG. 10 illustrates an exemplary configuration for reagent pack receiver64. In particular, FIG. 10 illustrates reagent pack receiver 64including a slot to receive reagent pack 56. As shown in FIG. 10, theslot may include septum piercing needles to puncture vessels of reagentpack 56 and allow the reagents to be routed to a multi-port valve 58and/or any valve respectively coupled thereto. The slot and piercingneedles allow for easy removal and installation while not requiring theoperator to make the fluidic connections. In some embodiments, it may beadvantageous to configure reagent pack receiver 64 to tilt back andforth. In particular, it may be advantageous, in some embodiments, toagitate one or more reagents within reagent pack 56. For example, it maybe advantageous to agitate particles in solution to reduce clumping in areagent pack vessel. Such agitation may be incorporated within reagentpack receiver 64 by tilting mechanism 72, various positions of which areillustrated in cross-sectional views of reagent pack receiver 64 inFIGS. 11A-C. In some embodiments, reagent pack 56 may include a smallair bubble within one or more of the reagent vessels to main suspensionof components within the respective reagents during oscillation oftilting mechanism 72. The presence of an air bubble may be particularlyadvantageous for reagents comprising particles to maintain theirsuspension within the accompanying slurry. In general, the operation oftitling mechanism 72 may be continuous, periodic, or sporadic.

Turning to FIGS. 12-15, flowcharts of exemplary methods for preparingfluid assays are shown. As noted above, the storage mediums of thesystems described herein may include program instructions which areexecutable by a processor for automating the preparation of a fluidassay, such as but not limited to the steps described in below inreference to the flowcharts depicted in FIGS. 12-15. Therefore, themethods described in reference to FIGS. 12-15 may be referred to as“computer-implemented methods”. It is noted that the terms “method” and“computer-implements method” may be used interchangeably herein. It isalso noted that the computer-implemented methods and programinstructions of the systems described herein may, in some cases, beconfigured to perform processes other than those associated with fluidassay preparation and, therefore, the computer-implemented methods andprogram instructions of systems described herein are not necessarilylimited to the depiction of FIGS. 12-15.

As shown in FIG. 12, a method for preparing a fluid assay may includeblock 80 in which a fluid sample is mixed with a first set of magneticparticles. In reference to system 51 of FIG. 12, the process of block 80may include infusing a fluid sample into input 53 and routing the fluidthrough multi-port valve 58 to reaction vessel 60. Subsequent orconcurrent thereto, a first set of magnetic particles from reagent pack56 may be routed to through multi-port valve 58 to reaction vessel 60 tomix with the fluid sample. The fluid sample may, in some embodiments, bepreprocessed (i.e. processed prior to being introduced into the systems)such as by the processing steps described below in reference to FIG. 13.In addition or alternatively, the state of the sample may be transformedprior to being introduced into the systems. For example, a solid sample,such as biological tissue, may be suspended within a buffer or an airsample may be condensed into a liquid. In other embodiments, the fluidsample may not be processed prior to being introduced into the systems.In such cases, system 51 may, in some embodiments, be configured toconduct some of the steps described below in reference to FIG. 13. Forexample, input 53 may, in some cases, include a filter. In addition oralternatively, reagent pack 56 may include a lysing agent for lysingcells within the fluid sample. In such cases, it may be particularlyadvantageous for system 51 to include a sonication system to insure thecells are lysed after a certain incubation time.

It is noted that other reagents which are known for processing a fluidsample may be additionally or alternatively included within reagent pack56 for mixing with the magnetic particles and the fluid sample duringblock 80, such as but not limited to those specific to processing tissueor fluid samples. Consequently, the methods and the systems describedherein are not necessarily restricted to the aforementioned processes.In any case, incorporating the aforementioned process steps into system51 can expand the functionality of process module 6 to perform twoprocesses: the automation of sample processing and the automation ofassay preparation. Sample processing is the conversion of a raw sampleinto a form that is compatible with the desired assay. Assay preparationtakes the converted sample and forms a particle based assay.

In general, the first set of magnetic particles referenced for mixingwith the fluid sample in block 80 may be configured to react with thefluid sample to capture a desired agent upon the magnetic particles. Forexample, in some cases, the first set of magnetic particles may beconfigured to capture nucleic acid from a fluid sample. Such a processis illustrated in the nucleic acid assay flowchart depicted in FIG. 14and is described in more detail below. Alternatively, the first set ofmagnetic particles may be configured to capture antigens located in abiological sample (such as tissue or bodily fluid). Such a process isillustrated in the immunoassay flowchart depicted in FIG. 15 and isdescribed in more detail below. It is noted that magnetic particles arereferenced herein as reagents and, therefore, may constitute a reagentfor which reagent pack 56 may be configured to store for the preparationof a fluid assay. More specifically, the term “reagent” as used hereinmay generally be referred to herein as a substance used to prepare aproduct because of its chemical or biological activity.

Subsequent to a predetermined incubation time (which may beassay-specific) for process described in block 80, the method maycontinue to block 81 in which the first set of magnetic particles areimmobilized with a magnetic field. Such a process may include moving anactuator to which one or more magnets of system 51 is coupled inproximity to reaction vessel 60. Subsequent thereto, the method maycontinue to block 82 in which the fluid is separated from the first setof magnetic particles. In particular, system 51 may be operated toremove unreacted fluid sample from reaction vessel 60. In someembodiments, the method may continue mixing different fluid reagentswith the first set of magnetic particles subsequent to the separation ofthe magnetic particles from the fluid sample as shown in block 84. Insuch cases, after mixing with the magnetic particles, the method mayreiterate the steps of immobilizing the magnetic particles to separatethe different fluid reagents therefrom. For example, in some cases, awashing solution may be mixed with the first set of magnetic particlesto remove any unreacted components of the fluid sample previously mixedwith the magnetic particles. In addition or alternatively, otherreagents may be mixed with the first set of magnetic particles to removecomponents desirable for analysis, such as for example nucleic acid fornucleic acid assays. In other embodiments, reagents may be mixed withthe first set of magnetic particles to add components to the magneticparticles for subsequent analysis, such as immunoassays, for example.

In either case, the first set of magnetic particles may, in someembodiments, be analyzed as shown by the path between blocks 82 and 89.In such cases, the process of block 89 may include moving the first setof magnetic particles to microfluidic analysis module 5. In otherembodiments, the method may alternatively mix the solution separatedfrom the first set of magnetic particles (discussed in reference toblock 82) with a second distinct set of magnetic particles as shown inblock 86 of the flowchart depicted in FIG. 12. For example, in someembodiments, nucleic acid separated from the first set of magneticparticles in a nucleic assay (as described in reference to block 82) maybe mixed with a PCR reagent to start a PCR process, which is describedin more detail below in reference to FIG. 14. In such a case, the methodmay continue to block 87 in which the fluid is separated from the secondset of magnetic particles, which may include the immobilization of thesecond set of magnetic particles and the removal of the residual fluid.In some cases, the immobilization of the second set of magneticparticles may be by the same magnet used to immobilize the first set ofmagnetic particles. In other embodiments, however, the second set ofmagnetic particles may be immobilized by a different magnet withinsystem 51. Subsequent thereto, the second set of magnetic particles maybe analyzed as shown by the path between blocks 52 and 89. Proceduresfor analyzing the second set of magnetic particles may be generallywithin the scope described for analyzing the first set of magneticparticles.

As noted above, FIG. 13 illustrates a flowchart of exemplary steps thatmay be used to process a fluid sample, either prior to or subsequent tobeing introduced into system 51. In particular, FIG. 13 outlines somedetermination which may be considered as to how a fluid sample isprocessed. For example, the flow chart includes block 90 in which adetermination of whether the collected sample needs to be concentrated.Examples of embodiments in which a sample may need to be concentrated iswhen the sample volume needs to be reduced and/or the concentration ofanalyte within the sample is expected to be too low. FIG. 13 furtherincludes block 92 in which a determination of whether the collectedsample needs to be filtered. A filtering process may be advantageous forremoving particles which are not of interest or may interfere with theanalysis of the sample. In addition to such processes, FIG. 13 includesblock 94 in which a determination of whether the assay to be preparedneeds the cells of the collected sample to be lysed such that materialwithin a cell can be accessed for analysis. As described above inreference to FIG. 12, the lysing process may be performed prior orsubsequent to mixing a fluid sample with a set of magnetic particles.Following the determinations of blocks 90, 92, and 94, the flow chartincludes block 96 in which a determination of what type of assay is tobe performed. The flowchart depicted in FIG. 13 outlines that a nucleicacid assay or an immunoassay (protein based) may be prepared. Flowchartsoutlining exemplary methods for both types of assays are depicted inFIGS. 14 and 15, respectively, and are described in more detail below.It is noted that the methods described in reference to FIGS. 14 and 15may be performed by any of the system configurations described herein.

As shown in block 100 in FIG. 14, preparation of a nucleic assay mayinclude capturing nucleic acid on to a carrier, such as a magneticparticle, which is or can be immobilized. Thereafter, the nucleic acidcarrier may be immobilized and the remaining sample discarded as shownin block 102. In some cases, the nucleic acid carriers may be washedafter discarding the sample. Although such a process is not depicted inFIG. 14, it is not necessarily omitted therefrom. In blocks 104 and 106,a determination of whether the nucleic acid needs to be separated fromthe carrier is made and, if applicable, the nucleic acid is separatedtherefrom. In such cases, the solution may also be heated to remove thenucleic acid from the particles and, consequently, system 51 may, insome embodiments, include auxiliary heaters. The processes for blocks100, 102, 104, and 106 may generally be performed by system 51 asdescribed above in reference to processing in reaction vessel 60. Afterblocks 104 and 106, the method continues to blocks 108 and 110 in whicha determination of whether a reverse transaction to convert RNA to DNAneeds to be conducted and, if applicable, is performed in block 110.

Thereafter, a determination of whether real time monitoring (analysis)is to be performed with DNA amplification as outlined in block 112. Ifthe determination is to go forward with real time monitoring, a PCRprocess is performed with a PCR solution which may be provided byLuminex Corporation of Austin, Tex. The PCR process is outlined in block114 and is formed concurrently with plurality of steps 116 foramplifying DNA, introducing reporter tags (e.g., PE) onto the particles,and analyzing the particles. If a determination is made to forego realtime monitoring, the PCR process is not performed, but plurality ofsteps 116 are performed and when the particles are ready for analysis,they are analyzed by microfluidic analysis module 5. In either case,analysis results may be displayed on display 2 as shown in block 119. Ingeneral, the aforementioned RNA to DNA reverse transaction process, thePCR process, and plurality of steps 116 may be performed by system 51 asdescribed above in reference to processing in reaction vessel 60. In thecase that system 51 is configured for multiple use operations, themethod may continue to block 118 to reset the fluid assay preparationsystem/module (APM) for a new sample.

FIG. 15 illustrates a flowchart of an exemplary process for preparing animmunoassay. As shown in FIG. 15, the method may include block 120 inwhich a fluid sample is mixed with magnetic particles having antibodiesattached thereto. After an assay-specific incubation period, themagnetic particles are immobilized and washed as noted in block 122 andsubsequently additional antibodies are added to the magnetic particlesas noted in block 124. Subsequent thereto, the method continues to block126 in which the magnetic particles are immobilized and washed again. Adetermination as to whether the antibodies need to be tagged is shown inblock 128 followed by the appropriate steps if applicable. Thereafter,the particles are sent to microfluidic analysis module 5 as shown inblock 130. In general, each of the process steps leading up to block 130(i.e., blocks 120, 122, 124, 126, and 128) may be performed by system 51as described above in reference to processing in reaction vessel 60. Inthe case that system 51 is configured for multiple use operations, themethod may include resetting the fluid assay preparation system/module(APM) for a new sample after block 130.

In addition to being used with processing module 6, the reagentcartridges described in reference to FIGS. 6-10 can also be used foracquiring a sample. For example, FIG. 16 illustrates a variant of thereaction cartridge that allows for direct sampling with the cartridge.The cartridge shown in FIG. 16 is configured to acquire a sample acrossblood collection volume 96 by pressing microneedle array 98 against apatient's skin. Sheath fluid may be introduced to the reaction vesselthrough sheath inlet 100, and the sample may be removed from thereaction vessel through sample outlet 102. Sheath inlet 100 and sampleoutlet 102 may have any suitable configuration known in the art. Thereaction cartridge may also include a removable seal 104, which may beconfigured as described herein. The sample flows from the microneedlearray into a holding chamber for extraction by the reagent cartridgereceiver, one embodiment of which is shown in FIG. 17. In particular,FIG. 17 shows the operation of a reaction cartridge with microneedles.In particular, the microneedle cartridge may be initially sealed asshown by microcartridge 106. Top seal 108 of the microcartridge isremoved to expose microneedles 110. The microneedle region of thecartridge is then pressed against the patient's skin. The microneedlecartridge is resealed after a sample is acquired, as shown bymicrocartridge 112. Then, the microneedle cartridge is placed inreceiver module 114 for analysis. Placing the cartridge into thereceiver actuates two needles 116 and 118 that pierce the cartridge'sseals. One needle injects sheath fluid into the cartridge while theother needle receives the sample and sheath fluid combined flow. Thecartridge shown in FIG. 16 could simply be a sample taking componentthat passes the sample onto a reaction cartridge like those mentionedabove or could contain reagents to process the sample internally.

It is often the case that reagents used in biological detection must berefrigerated or frozen when stored. The embodiments described herein, ifused with such reagents, may include a refrigeration unit onboardportable system 12 for reagent storage module 4. One embodiment of areagent storage module is shown in FIG. 18, which can store andrefrigerate reagents and provide an interlock and indicator system sothat used reagents are not accidentally used more than once. Forexample, as shown in FIG. 18, the reagent storage module may includedetector reagent storage 120, which may have any suitable configurationknown in the art. In FIG. 18, the reagent storage module is shown withcartridges 122 removed (on left) and with cartridges 122 inserted (onright). In addition, the reagent storage module may include indicatorpanel 124 that includes one or more indicators for each reactioncartridge. For example, the indicator panel may include indicator 126for each reaction cartridge. The indicator may be changed depending onthe status of the reagent cartridges in the reagent storage module. Forexample, a red light may indicate a used cartridge, an unlit indicatormay indicate a missing reagent cartridge, and a green indicator mayindicate a new reagent cartridge. Although the reagent storage moduleshown in FIG. 18 is configured for storing a particular number ofreaction cartridges, it is to be understood that the reagent storagemodule may be configured to store any suitable number of reactioncartridges.

The embodiments described herein provide a number of advantages overother systems configured to perform measurements of samples. Forexample, the embodiments described herein are substantially insensitiveto their environments. In addition, the embodiments described herein arenot time consuming to manufacture. Additionally, the embodimentsdescribed herein are relatively simple to operate and can be used byrelatively untrained users. Furthermore, the embodiments describedherein are relatively inexpensive and are capable of performing themeasurements without requiring significant laboratory resources forpre-processing of the one or more samples. Moreover, the embodimentsdescribed herein are relatively flexible in design such that a singlesystem can be used for substantially different applications.

Further modifications and alternative embodiments of various aspects ofthe invention may be apparent to those skilled in the art in view ofthis description. Accordingly, this description is to be construed asillustrative only and is for the purpose of teaching those skilled inthe art the general manner of carrying out the invention. It is to beunderstood that the forms of the invention shown and described hereinare to be taken as the presently preferred embodiments. Elements andmaterials may be substituted for those illustrated and described herein,parts and processes may be reversed, and certain features of theinvention may be utilized independently, all as would be apparent to oneskilled in the art after having the benefit of this description of theinvention. Changes may be made in the elements described herein withoutdeparting from the spirit and scope of the invention as described in thefollowing claims.

1-28. (canceled)
 29. A chip-based flow cytometer comprising: (a) asubstrate having fixedly arranged therein: (i) a first input conduit forreceiving a sample fluid containing fluorescent particles; (ii) a secondinput conduit for receiving a sheath fluid; (iii) a fluid flow chambercoupled to the first and second input conduits, wherein the fluid flowchamber is configured to generate a fluid stream with the sample fluidconfined within the sheath fluid; (iv) a channel coupled to the fluidflow chamber for routing the fluid stream through an opticalinterrogation region; and (v) an outlet coupled to the channelconfigured for the sample fluid and the sheath fluid to exit thesubstrate after passing through the optical interrogation region; (b) anillumination system configured to direct light at a plurality ofdifferent spots within the optical interrogation region of the channel;and (c) a collection system comprising a plurality of detectorsconfigured to gather fluorescent light emitted from the fluorescentparticles in the optical interrogation region, wherein the fluorescentlight emitted from the fluorescent particles at each of the differentspots is collected by a different detector of the collection system. 30.The chip-based flow cytometer of claim 29, wherein the illuminationsystem comprises a light source and a beam splitter.
 31. The chip-basedflow cytometer of claim 29, wherein the light source is a light emittingdiode.
 32. The chip-based flow cytometer of claim 29, wherein theillumination system comprises a different light source for each of thedifferent spots within the optical interrogation region.
 33. Thechip-based flow cytometer of claim 31, wherein at least one of the lightsources emits light within a different wavelength range than another ofthe light sources.
 34. The chip-based flow cytometer of claim 29,wherein the plurality of detectors comprises a silicon avalanchephotodiode.
 35. The chip-based flow cytometer of claim 29, wherein thefluorescent particles are beads.
 36. The chip-based flow cytometer ofclaim 29, wherein the fluorescent particles are cells.